BRUCELLOSIS (3)

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BRUCELLOSIS : 

BRUCELLOSIS

BRUCELLOSIS : 

It is term used as a convenient description for all phases of the disease caused by a bacteria called Brucella. Many names have been applied to it as: 1. Malta fever 2. Mediterranean fever. 3. Gibraltar or rock fever 4. Undulant fever. BRUCELLOSIS

Brucellosis overview : 

1. Introduction and history 2. Epidemiology 3. Sources of transmission 4. Taxonomy 5. Morphology 6. Cultural characteristics 7. Antigenic structure 8. Biochemical reactions and classification 9. Variation 10. Virulence 11. Clinical spectrum 12. Diagnosis 13. Prophylaxis 14. Treatment. Brucellosis overview

Introduction and history : 

1887 – Bruce reported small coccal organisms in the stained sections of spleen from a fatally infected soldier. he named this as Micrococcus melitensis. 1897 -- Wright and Smith – agglutinatiuon test for detecting antibodies to M.melitensis in the serum of humans and animals. 1918 – Alice C Evans drew attention to the similarity between M.melitensis and organism described by Bang (1897) as the cause of contagious abortion of cattle. Introduction and history

Slide 5: 

1920 – Meyer and Shaw proposed the name Brucella for the two organisms in the honour of Sir David Bruce. NATURAL HOSTS : B.melitensis -- sheep and goats. tends to localise in the reticulo- endothelial system and genital tract. most severe and acute human infection. B.abortus -- cattle. less virulent than B.melitensis. can also cause Brucellosis in humans,but in milder form. B.suis -- discovered by Tram in 1914. natural parasite of pigs. also transmissible to humans. chronic suppurative lesions in skeletal system .

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B.canis -- reported by Carmichiel and Bruner(1968). as cause of abortion among dogs in U.S.A. occasional cases in humans. B.ovis -- first observed in Australia and New Zealand by Buddle & Boyes , Simmons & Hall in 1953 as the cause of sexually transmitted epididymitis in rams. In humans -- no confirmed reports B. neotomae -- isolated by Stoenner & Lackman in 1957 from desert wood rats. not been associated with disease in humans.

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EPIDEMIOLOGY Most cases of human brucellosis ----- B.melitensis In Europe --. 1. Mediterranean region 2. southern edges of Medittereanean base like Egypt. Libya, Isreal, Lebanon, Syria. Brucellosis is also common in many parts of North, central and south America purticularly in Mexico, Argentina, Brazil, Colombia and Peru. B.melitensis is common in some areas of Indian Sub continent.

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B.abortus -- World wide B.suis -- Endemic in southern US, South East asia and Latin America. B.canis -- Latin America. Central Europe and Japan.

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INDIAN SENARIO Species of main concern in India are B.melitensis, and B. abortus. B. melitensis is the most virulent and common strain for man and it causes severe and prolonged disease with a risk of disability. B. abortus is the dominant species in cattle. In a separate study carried out by Mathur (1968) in Haryana, concluded the goats and sheep as the sources of human infection by isolating B. melitensis as a predominant strain from human blood as well as milk samples from goats and sheep. As many as 4.2% aborted women were seropositive for the disease (Randhawa et al 1974).

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Sen et al (2002), identified 28(6.8%) seropositive cases in a group of 414 patients with PUO and Kadri et al (2000), identifi ed 28(0.8%) seropositive cases in a group of 3,532 patients with PUO.

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Brucellosis -- ZOONOSIS Human infection -- direct or indirect contact with infected animal tissue. Person to person transmission -- RARE in circumstances implicating sexual contact , tissue transfer including blood and bone marrow. Laboratory acquired Brucellosis -- accidental ingestion, inhalation, injection, mucosal and skin contamination. Exposure to infectious aerosols during manipulation of cultures is one of the most common source of laboratory infection.

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In Britain , the advisory group on dangerous pathogens has classified Brucella as a pathogen requiring containment at the levelof category -3 Mainly Farmers, abattoir workers, butchers, veterinarians are at risk.infection can occur through contamination of conjunctiva and skin with discharges Main source of infection to general population is by dairy products prepared from infected milk. Neonatal infection can be acquired by the transplacental route, during delivery or via the ingestion of contaminated breast milk.

Taxonomy : 

Molecular genetic studies (ie, restriction endonuclease mapping) revealed gene poly morphisms that were able to differentiate species of brucella. One of the poymorphic gene that help to differentiate species is omp2 porin gene, which codes for a 36 –kDa OMP that is resposible for determining the susceptibility to the dyes used for conventional species identification. Unlike other bacteria Brucella species have two chromosomes except for B.suis biovar 3, which has a single chromosome. The genome of B.melitensis strain 16M was recently sequenced and is composed of 32,94,935 base pairs(bp) distributed over two circular chromosomes of 21,17,144 bp and 11,17,787 bp respectively. Taxonomy

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Brucella species are now classified in the α2 sub group of the Proteobacteria in the family Brucellaceae in the proposed order “Rhizobiales” MORPHOLOGY small round or oval coccobacilli. 0.4 μm in diameter ,but bacilli are 1-2 μm in length . arranged singly , in pairs ,short chains or small clusters.

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Donot produce capsule,spores or flagella. Gram negative, non acid fast. Modified Ziehl-neelsen stain is used for screening placentas and other products of abortion. Brucella cells have a red or orange appearance; other patogens like Coxiella burnetti and chlamydiae have a similar appearance.

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Cultural characteristics: Most Brucella strains require multiple amino acids, thaimine, biotin, picotinamide, and pentothenic acid, Isoerythritol for growth. Grow poorly on peptone media, but enhanced growth is seen by the addition of serum ,blood and tissue extracts. Optimum PH range is 6.6 – 7.4. Temperature range is 20 -40 ⁰ C, optimum is 37⁰ C. Strictly aerobic but some strains of B.abortus require the addition of 5-10 % CO2..

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serum dextrose agar – raised, convex, circular, translusent colonies, 0.5- 1mm in diameter,after incubation for 48 hrs at 37⁰C. Brucella like Gram negative bacterium contain LPS in the outer cell membrane of the cell wall. But structurally and functionally different from G-ve bacteria. The lipid A part of the LPS contains 16 – carbon long fatty acids but lacks 14 carbon myristic acid typical of lipid A of Enterobacteriaceae. On the basis of the Polysaccharide O portion of LPS somatic antigens of Brucella are classified into two major types : antigen A and antigen M.

Slide 19: 

variation : Typical virulent brucella -- colonies are smooth and translucent. Brucella species undergo antigenic variation or dissociation on subculture. colonies switch from a smooth to rough morphology. On the molecular level this antigenic variation is the result of decreased expression of genes encoding the additional glycosylation of the polysaccharide moieties of the cell wall lipopolysaccharide.

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Non smooth variants -- -intermediate (I), rough (R), mucoid (M) - show loss of virulence. -R and M strains will not agglutinate with - homogenous antisera prepared against - smooth strains B.abortus ,B.melitensis, B.suis -- usually produce smooth colonies on primary isolation. B.canis --- invariably non smooth.

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Isolation of Brucella from contaminated material Requires a selective media : this contains bacitracin, nilidixic acid, cycloheximide, nystatin, polymyxin B, and vancomycin in a serum dextrose agar base. A liquid medium ( Santon & Brodie) -- addition of Amphotericin B, D-cycloserine . -- suppression of contaminants.

Slide 22: 

For isolation of brucella from human blood and bone marrow samples -- two phase culture system of the type devised by Castaneda (1947) is recommended. Sensitivity of the isolation can be improved -- lysis – concentration method for processing the samples. Use of rapid , automated detection systems -- reduce the time taken to detect positive cultures. Culture - incubated in an atmosphere containing 5-10 % CO2. - Retained upto 6 weeks as growth may be slow, purticularly if the patient has received previous antibiotics.

Slide 23: 

Biochemical reactions: Brucella -- 1. catalase & oxidase positive (except B.ovis, B.neotomae oxidase negative). 2. Reduce nitrate to nitrites. 3. variable urease activity B.suis [U+] -- 30 min B.abortus [U+] – 1-2 hrs 4. some produce H2S , some donot. 5. citrate – not utilised 6. MR and VP test - negative 7. indole - not produced 8. Do not ordinarily ferment any sugars.

Slide 24: 

Susceptibility to physical and chemical agents : 1. Rapidly killed at 60 C in 10 min. Hence killed by pasteurization in the milk. 2. Killed by disinfectants like 1% phenol in 15 min. 3. Sensitive to direct sunlight and acid. Brucella survive - refrigerated milk - 10 days ice cream - 1 month butter - 4 months B.melitensis - urine - 6 days dust - 6 weeks water or soil - 10 weeks

classification : 

Brucella may be categorized into species and biovars by the following tests. For these suspension of test strain is prepared in 1.5 ml of 0.85% sterile nacl to give c. 10 8 colony forming units /ml. 1. CO2 requirement 2. H2S production 3.Growth in the presence of aniline dyes like basic fuchsin and thionine. 4.Ability to use glutamic acid, ornithine, lysine and ribose 5. Agglutination with antisera against specific (LPS). 6. Susceptibility to lysis by specific bacteriophage. classification

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Virulence Organisms that are in the smooth phase possess a smooth - type LPS (S-LPS) and are resistant to intracellular killing by polymorphonuclear Cells, by inhibiting lysosomal degranulation and the respiratory burst associated with PMN activation. The LPS of B.abortus, B.melitensis, B.suis has two major antigenic determinants called A (for “Abortus”) and M ( for “Melitensis”). They play a role in organism virulence. Serodominant A antigen -- rod shaped Serodominant M antigen -- kinked in shape.

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Phage Lysis : Tbilisi (Tb) is the reference phage used for Brucella typing. This phage lyses B.abortus both - Routine test dilution and 10,000 RTD. B.suis - at 10,000 RTD. Only Do not lyse any strain of B.melitensis.

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The common expression of nonterminal α 1,2 linked N-formyl-D-perosamine is responsible for the cross reactivity seen between S-LPS of smoooth B.abortus and smooth B.melitensis strains and the cross reactivity with other species like Vibrio cholerae 0:1, Yersinia enterocolitica 0:9 , Escherichia coli 0:157, Salmonella 0;30 and Stenotrophomonas maltophilia. B.canis and B.ovis -- do not have A & M antigenic determinants. characteristically rough and do not display phase variation. .

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PATHOGENESIS Inside the host 1. AMP & GMP mediated phagocytized but able to survive -- inactivation of intracellular myeloperoxidase-peroxide defence mech. 2. production of SUPEROXIDE DISMUTASE which blocks the formation of toxic O2 radicals carried to lymph nodes, blood stream, reticuloendothelial system like liver, spleen, and bone marrow. PMNs degenerate and release intracellular organisms Again endocytosed by macrophages and monocytes

Slide 31: 

The “undulant” waxing- and- wanning fever pattern seen in brucellosis is associated with the periodic release of bacteria and their components from phagocytic cells. TNF α , TNF γ , IL-1, IL-2 influence macrophage mediated anti brucella activity during cell mediated immune response. Brucella synthesize and release a high M W protein factor that inhibits TNF α expression in activated macrophages. This inhibition likely to contribute to the ability of Brucella species to evade human host defences and maintain intracellular existence.

Clinical spectrum : 

Incubation period : 2 – 3 weeks ( range 1 week to 2-3 months) Symptoms may be abrupt or develop over a period of days to months Non specific symptoms : fever , night sweats , chills, malaise, headache, myalgias , arthralgias. Lymphadenopathy, Splenomegaly , Hepatomegaly may also be seen. Cutaneous manifestations : erythema nodosum like lesions, maculopapular rash, DVT may be noted in some patients. Clinical spectrum

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Osteo articular infections: Arthritis, Sacroilitis - children Spondylitis, Vertebral osteomyelitis, para vertebral abscess - chronic infection in older pts. CNS infection : ccurs in less than 5% presents as meningitis, encephalitis, meningomyelitis. patients have CSF pleocytosis, protein , low – normal glucose. Culture of brucella from CSF is negative more than 75% of time although blood cultures may be positive.

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Pulmonary infections : result of hematogenous spread or by direct inhalation. Presents as bronchitis, bronchopneumonia, lung abscess, hilar lymphadenopathy, pleural effusion, empyema. GIT & Hepatobiliary tract infection: seen in 70% of patients . Abdominal pain , nausea, vomiting, diarrhea or constipation. long standing cases : colitis, enterocolitis, spontaneous bacterial peritonitis. liver involvement : caseating granulomas – B.melitensis, B.suis non caseating granulomas – B.abortus.

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GUT infection: epididymitis, prostatitis, orchitis, and renal granulomas. Brucella cause abortion in animals by localization in the chorioamniotic membranes of the placenta, but very little evidence of the same in humans. CVS infection : seen in less than 2%. Brucella endocarditis is the main cause of death. Aortic valve involvement occurs most frequently. Ocular infection : late complications like optic nueritis, keratitis, endopthalmitis. positive cultures can be obtained from aqueous and vitreous humors.

Laboratory diagnosis : 

Culture : Isolation of Brucella sp. is the most certain for diagnosis . samples of any body fluid, or tissue can be cultured. blood – most common. bone marrow - gives more positive results. Blood is collected -- pyrexial phase 3-5 ml inoculated -- into each of 4 blood culture bottles having 20-100 ml of serum dextrose broth, on 3 successive days. Incubated at 37 ⁰ C in air enriched with 5-10 % CO 2 After 4 th day of incubation subcultured - twice a week for 8 weeks on to SD agar. (Serum dextrose agar ) Laboratory diagnosis

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If anti coagulant needed – trisodium citrate, preservative free heparin be used Blood clot may also be cultured but it should be homogenized first. Blood cultures -- 8 weeks before they are discarded as negative. Frequent subculture can be avoided -- Casteneda biphasic culture technique in which both solid and liquid media of trypticase soy agar and broth respectively are contained in one single bottle. For subculture bottle is tilted so that that the broth flows on to solid medium and again kept in upright position . Even in this subculture is done -- when growth appears - to minimise dissociation from smooth – nonsmooth colonial phase.

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Isolation rate -- improved by -- lysis concentration method. Bactec, BACT/ALERT systems -- sensitivity when combined with lysis concentration method. PCR -- using 223 bp region of the 31 kDa surface protein of B.abortus On Mair’s medium (gentian violet) -- blue to violet with black centre. SD agar -- pale yellow - translucent

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Serum dextrose agar : Davies new zealand agar - 15 g Bacteriological peptone - 10 g Lab-lemco extract - 5g Nacl - 15g Distilled water - 1 litre. pH adjusted to - 7.5 Sterilised at 121 C - 20 min Enrich the base with - 1% sterile glucose & 5 % inactivated horse serum before dispensing into petri dishes, tubes for slants.

Serological tests : 

when the infecting organism not isolated from blood or other clinical specimen - serological investigation (sub clinical and for acute and chronic cases) becomes paramount important. serum samples are collected as soon as possible & at various stages of the disease. most widely used tests - Standard agglutination test (SAT) Complement fixation test (CFT) Indirect immunofluorescence others - mercaptoethanol agglutination test(ME) Radio immune assay (RIA) ELISA Serological tests

Slide 41: 

IgM and IgG antibodies - appear after 7-10 days of infection. IgM persist - 3 months thereafter decline. IgG and IgA - appear after 3 weeks of infection Rising titers of antibodies by SAT can be a good help in confirming the diagnosis.

Standard agglutination test (SAT) : 

Described by Robertson et al (1980). Detects either IgM or IgG. Most commonly used serological test. ( tube agglutination test ) Detect antibodies - O- polysachharide (LPS) component of Brucella. Antigen used - killed strains of B.abortus. Used for diagnosis of - B.abortus , B.melitensis, B.suis. Not useful for B.canis - no O polysachharide - no antibodies against LPS Tube agglutination test - positive - antibody titres ≥ 1 in 160. (or) - 4 fold rise titer in convalescent sera. Standard agglutination test (SAT)

Modified tube agglutination test : 

1. In this , 2 mercaptoethanol is added to patients sera before testing. 2. Mercaptoethanol disrupts - disulfide bonds of IgM . Hence only IgG is detected 3.Useful for specific detection of IgG and titers higher than 1:80 are suggestive of active infection. 4. High IgG antibody titer or a titer that is higher after treatment suggests relapse or persistent infecton. 5.useful for diagnosing Brucellosis during convalescence. Modified tube agglutination test

Guinea pig inoculation : 

Guinea pig - most susceptible to brucella. Infection - non progressive and may recover spontaneously Inoculation - into thigh Examination - animal is killed after 6-8 weeks. Lymph node in the region of inoculation may be enlarged and should be cultured. Spleen - should be cultured by rubbing the cut surface on the surface of SD agar containing bacitracin 10 units/ml , polymyxin B 4 uints/ml to suppress the contaminants. Blood from JV - tested for presence of antibodies to brucella Guinea pig inoculation

INDIRECT IMMUNOFLUORESCENCE : 

Sensitive method to detect brucella antibodies even in agglutination negative cases. ELISA Most sensitive for detection of IgM, IgG, IgA brucella antibodies. In this test cytoplasmic depleted antigen of brucella is used as antigen. BRUCELLA SKIN TEST : 1. delayed type of hypersentivity reaction. 2. protein extract - antigen - Intradermally. 3. Induration ≥ 6 mm - 24 hrs - Positive. 4. Positive only in chronic cases but negative in acute. 5. Repeated negative skin tests excludes Brucellosis INDIRECT IMMUNOFLUORESCENCE

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Nevertheless serological tests show errors due to following: 1.Blocking Antibodies : 1 .These are non agglutinating antibodies, 2. Gives false results. 3. Avoided by heating the serum at 55 C for 30 min 4 . By using 4 % saline or diluent in the test. 2. Prozone phenomenon: 1. Due to high levels of brucella antibodies in the serum. 2. Avoided by routine dilution of serum to atleast 1:320 because inhibition of agglutination may occur at low dilutions. 3. Cross reactivity : with Vibrio cholerae, Yersenia enterocolitica serotype 09, Francisella tularensis and Salmonella species due to cross reacting LPS in these.

Laboratory diagnosis in animals : 

1.culture of milk and urine from infected animals gives positive results. Rapid diagnostic methods : Rapid latex agglutinating test. Rose Bengal Card test. serological test : Milk Ring Test - To demonstrate antibodies in the milk of the animal. Conc suspension of killed B.abortus or B.melitensis stained with hematoxylin is used as antigen. Add a drop of colored antigen to the milk sample in a test tube and mixed well and incubated in water bath at 70 C for 40 -50 min. positive test - blue ring at the top leaving the milk unstained negative test - whole milk remains uniformly blue. Laboratory diagnosis in animals

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Treatment : Successful treatment for brucella - Prolonged treatment . Combination of anti microbials. Traditional regimen : Oral Tetracyclines - 6 weeks + IM Streptomycin / Gentamycin daily – 2-3 weeks. Most effective for most forms : Doxycycline 200 mg / day + Streptomycin 1g /day IM for 2-3 weeks others : doxycycline + rifampicin - 6 weeks orally

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Neuro brucellosis Doxycycline Brucella endocarditis -- + Rifampicin + Trimethoprim- sulfamethaxazole Prevention and control: 1. Pasteurisation and boiling of milk. 2. use of protective clothing and gloves. Vaccination of animals : 1. effective 2. live attentuated B.abortus 1019 strain is used. No vaccine is available for humans.

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